The Screening Of MicroRNA Related To T Lymphocyte Activition, And Its Target Were Prediction,Constraction And Identification

microRNA referred to as miRNA, is 21-25 base pairs of non-coding small molecule RNA, that it exists in a wide range of eukaryotes. miRNA gene transcription product of the primary (pri-miRNA) in the nucleus has been cut RNaseâ…¢Drosha to become precursor miRNA (pre-miRNA), pre-miRNA in the transporter protein of the role of exportin-5 by the nucleus to the cytoplasm, the and then by a further cut RNaseâ…¢Dicer have mature miRNA. These mature miRNA components and other proteins with RISC (RNA-induced silencing complex) complex, which led to the degradation of target mRNA or translation inhibition. miRNA widely exists in animals, plants and virus, the first was confirmed that the online pest first discovered in lin-4 and let-7,followed by a number of research team, including humans, fruit flies, plants and other biological species identified hundreds of miRNAs. As of September 2008, available on the Sanger miRNA data miRBase12.0 (http://microrna.sanger.ac.uk/sequences/) of the miRNA in the tot al number of 8 273. Large number of studies show that, miRNA involved in cell division, including proliferation, differentiation and development, as well as metabolism and many other important biological processes.Mammalian immune system in resistance to infection, their stability and immune surveillance play an important role. In order to achieve the immune response and the appropriate balance between immune tolerance, immune response initiation, termination and intensity of reaction must be strict regulation. Transcription factor in relation to full-or all-way clearance adjustment, miRNAs are also suitable for fine regulation of the immune system and quantitative regulation, because they adopted the role of a very short period of regulated sites, the role of sites in one or a few base pairs will be able to play a key role, and because they are easy to process in the evolution of mutation, so as to provide the following pathogenic organisms at the same time play a role in the evolution of the gene regulator repository. Recent studies indicate that miRNAs indeed fine-tune the immune system play an important role. MiRNA-depth understanding of the mechanisms regulating the immune system not only helps to regulate the immune response to identify new targets, and contribute to the establishment of effective new therapies miRNA.In this experiment, in order to explore whether T cell activation have an impact on the expression pattern of miRNA, we applied cDNA microarray Exiqon miRNA to the activation of Jurkat cells (using CD3 antibody and CD28 antibody two-signal activation) for the experimental group, no activation Jurkat cells as a control group, analysis of the two differences in miRNA expression profiling to identify significant changes in expression levels (more than 2-fold reduction) of the miRNA. The results showed that the chip, and is not activated Jurkat cells compared to Jurkat cells activated a number of miRNA expression levels were significantly reduced. We are part of the miRNA down a real-time quantitative PCR testing to verify the results of the chip.In order to really miRNA and human T lymphocyte activation, we adopted miRanda to predict miRNA in T cell activation potential role in the process of site, and on this basis to verify. The results show that the forecast: miR-33b and miR-568 in the process of the role of many important elements have sites of these molecules in T cell activation, including CD96, CD28, NF1, CD3G, IL2RA, IL2RB, NFAT5, CD8B, RAC3, etc.. Which, miR-568 target of NFAT5 in predicting the score of the target, the highest scores, and miR-568 and NFAT5 has the potential role of four sites that NFAT5 is likely that miR-568 against a target .In order to verify these predicted miRNA target is a real target, we will forecast the 3'UTR sequence of the target miRNA binding site in the upstream and downstream part of fragment into pGL3-Control-MCS2 luciferase expression vector, the Construction of dual luciferase reporter system, and then, respectively, of eukaryotic expression plasmid miRNA or miRNA, as well as synthetic or synthetic pcDNA3 control of negative control scramble miRNA (NC) and the construction of a good target eukaryotic expression vector, pRL-TK plasmid cotransfected HEK293 cells, luciferase activity observed changes. The results showed that: miR-568 on a relatively NFAT5 inhibited, suggesting that miR-568 may NFAT5 activation of T cells play a role.